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A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

Bhutta MS, Roy B, Gould GW, McInerny CJ - PLoS ONE (2014)

Bottom Line: Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic.Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase.Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

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ESCRT proteins are required for septation in fission yeast.(a) Defective septation in fission yeast strains containing chromosomal deletions of ESCRT genes. Wild-type and strains containing individual chromosomal deletions of ESCRT genes were grown at 25°C in complete liquid medium to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Panels A-F show representative cells illustrating observed septation phenotypes. Schematic diagrams above panels represent each phenotype: (A) a normal septum, (B) a misaligned septum, (C) a non-perpendicular septum, (D) multiple septa, (E) no septal formation, and (F) failed separation of daughter cells following septation. Scale bars, 10 µm. Data from a typical experiment repeated three times is shown. (b) Quantitative analysis of the frequency of septation phenotypes A–F in strains containing ESCRT chromosomal deletions, in comparison to wild-type. In each case, 400 cells were counted in triplicate. An asterisk (*) indicates a p value<0.05, indicating a significant difference to wild-type; n = 3. Each of the ESCRT gene labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).
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pone-0111789-g001: ESCRT proteins are required for septation in fission yeast.(a) Defective septation in fission yeast strains containing chromosomal deletions of ESCRT genes. Wild-type and strains containing individual chromosomal deletions of ESCRT genes were grown at 25°C in complete liquid medium to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Panels A-F show representative cells illustrating observed septation phenotypes. Schematic diagrams above panels represent each phenotype: (A) a normal septum, (B) a misaligned septum, (C) a non-perpendicular septum, (D) multiple septa, (E) no septal formation, and (F) failed separation of daughter cells following septation. Scale bars, 10 µm. Data from a typical experiment repeated three times is shown. (b) Quantitative analysis of the frequency of septation phenotypes A–F in strains containing ESCRT chromosomal deletions, in comparison to wild-type. In each case, 400 cells were counted in triplicate. An asterisk (*) indicates a p value<0.05, indicating a significant difference to wild-type; n = 3. Each of the ESCRT gene labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).

Mentions: Microscopic visualisation of dividing cells in wild-type and ESCRT deletion mutant strains revealed striking differences in their respective septation indices (Fig. 1). An asynchronously dividing population of wild-type cells displayed ∼87% containing normal septa (Class A) and ∼12% separating cells (Class F). Deletion mutants of genes encoding components of the various classes of ESCRT proteins were viable [14]. Significantly different numbers of cells showing the Class F phenotype were displayed in sst4Δ (∼42%, ESCRT-0), vps20Δ (∼46%), vps24Δ (∼45%, ESCRT-III) and vps4Δ (∼45%) cells, suggesting that the timing of separation in these cells was affected (Fig. 1b). Furthermore, sst4Δ (ESCRT-0), sst6Δ and vps28Δ (ESCRT-I), vps25Δ (ESCRT-II), vps20Δ, vps24Δ and vps2Δ (ESCRT-III), and vps4Δ cells displayed additional defective septal phenotypes (Classes B–D), suggesting that septal formation had occurred incorrectly. Some mutants also showed the absence of septa in cells of a length that would be expected to contain them (Class E), such as sst6Δ, vps28Δ, vps25Δ, vps24Δ and vps4Δ, which suggested that septal formation was delayed. Similar observations were made at 30°C (Fig. S1). Collectively, these observations demonstrate that septal formation is affected by the absence of various classes of ESCRT proteins in fission yeast, implying that they contribute to cell separation in this organism.


A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

Bhutta MS, Roy B, Gould GW, McInerny CJ - PLoS ONE (2014)

ESCRT proteins are required for septation in fission yeast.(a) Defective septation in fission yeast strains containing chromosomal deletions of ESCRT genes. Wild-type and strains containing individual chromosomal deletions of ESCRT genes were grown at 25°C in complete liquid medium to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Panels A-F show representative cells illustrating observed septation phenotypes. Schematic diagrams above panels represent each phenotype: (A) a normal septum, (B) a misaligned septum, (C) a non-perpendicular septum, (D) multiple septa, (E) no septal formation, and (F) failed separation of daughter cells following septation. Scale bars, 10 µm. Data from a typical experiment repeated three times is shown. (b) Quantitative analysis of the frequency of septation phenotypes A–F in strains containing ESCRT chromosomal deletions, in comparison to wild-type. In each case, 400 cells were counted in triplicate. An asterisk (*) indicates a p value<0.05, indicating a significant difference to wild-type; n = 3. Each of the ESCRT gene labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214795&req=5

pone-0111789-g001: ESCRT proteins are required for septation in fission yeast.(a) Defective septation in fission yeast strains containing chromosomal deletions of ESCRT genes. Wild-type and strains containing individual chromosomal deletions of ESCRT genes were grown at 25°C in complete liquid medium to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Panels A-F show representative cells illustrating observed septation phenotypes. Schematic diagrams above panels represent each phenotype: (A) a normal septum, (B) a misaligned septum, (C) a non-perpendicular septum, (D) multiple septa, (E) no septal formation, and (F) failed separation of daughter cells following septation. Scale bars, 10 µm. Data from a typical experiment repeated three times is shown. (b) Quantitative analysis of the frequency of septation phenotypes A–F in strains containing ESCRT chromosomal deletions, in comparison to wild-type. In each case, 400 cells were counted in triplicate. An asterisk (*) indicates a p value<0.05, indicating a significant difference to wild-type; n = 3. Each of the ESCRT gene labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).
Mentions: Microscopic visualisation of dividing cells in wild-type and ESCRT deletion mutant strains revealed striking differences in their respective septation indices (Fig. 1). An asynchronously dividing population of wild-type cells displayed ∼87% containing normal septa (Class A) and ∼12% separating cells (Class F). Deletion mutants of genes encoding components of the various classes of ESCRT proteins were viable [14]. Significantly different numbers of cells showing the Class F phenotype were displayed in sst4Δ (∼42%, ESCRT-0), vps20Δ (∼46%), vps24Δ (∼45%, ESCRT-III) and vps4Δ (∼45%) cells, suggesting that the timing of separation in these cells was affected (Fig. 1b). Furthermore, sst4Δ (ESCRT-0), sst6Δ and vps28Δ (ESCRT-I), vps25Δ (ESCRT-II), vps20Δ, vps24Δ and vps2Δ (ESCRT-III), and vps4Δ cells displayed additional defective septal phenotypes (Classes B–D), suggesting that septal formation had occurred incorrectly. Some mutants also showed the absence of septa in cells of a length that would be expected to contain them (Class E), such as sst6Δ, vps28Δ, vps25Δ, vps24Δ and vps4Δ, which suggested that septal formation was delayed. Similar observations were made at 30°C (Fig. S1). Collectively, these observations demonstrate that septal formation is affected by the absence of various classes of ESCRT proteins in fission yeast, implying that they contribute to cell separation in this organism.

Bottom Line: Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic.Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase.Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

Show MeSH
Related in: MedlinePlus